How to turn a mint into catmint: the origins of specialised metabolism

As they cannot move, plants must find innovative ways to defend themselves and interact with their environment. The solution that many plants have come up with is to make unique chemicals with a particular function, such as repelling insects or providing protection from the sun's UV rays. Many of these chemicals, known as natural products, are used by humankind for fragrances and medicines. Finding out how plants have evolved to make these chemicals will help us understand a core aspect of how plants adapt to the environment. It may also provide us with lessons for how to make similar natural product-like chemicals in the future.

We are investigating how plants evolve to make new chemicals. Our research focuses on catmint, also known as catnip or Nepeta. Catmint is famous for its euphoric effects on cats, which is the result of a chemical produced by catmint called nepetalactone. Catmint is part of the mint family, which includes plants such as lavender, oregano and, of course, mint. However, catmint has evolved uniquely amongst the mints to make nepetalactone. We will try to find out exactly how a mint has evolved into catmint: how did Nepeta evolve to make this unique compound?

To investigate this we will first obtain genome sequences of different catmint species and close relatives. A genome sequence is essentially a collection of an organism's genes, and is often considered a DNA "blueprint" or a set of instructions for an organism. The genomes will show us the genes that are involved making nepetalactone in the different species, and we can also see how genes are positioned on the genome relative to each other. We can perform calculations to estimate what the genes of catmint ancestors looked like many millions of years ago. This allows us to resurrect and test these ancient genes in the lab to find out what chemicals the ancestors of catmint could make.

Once we have enough genomes to perform good and robust analyses, we will focus on genes called NEPS that are responsible for making nepetalactone. These genes went through remarkable rapid changes in the ancestor of catmint. We will resurrect these ancient genes at important time points in catmint evolutionary history and test them in the lab to see how their behaviour has changed. We will be looking out for what changes occurred for them to transform from a normal gene to one that can make nepetalactone.

However, plants need many genes to make chemicals like nepetalactone, not just one. We must therefore extend this ancestral analysis to multiple genes, and we plan to test a variety of genes simultaneously to recreate an "ancestral metabolism" and so determine what chemicals the ancestors of catmint could make. We will also try to work out where in the genomes the genes were positioned during evolution. Genes appear to jump around in genomes as they evolve and we hope to discover why that happens by comparing the gene's behaviours with the genome locations.

Conducting all of this research will allow us to answer when and how catmint gained the ability to make nepetalactone. This, in turn, will provide wider lessons for how plants make new chemicals and how their genes and genome evolve to adapt to the environment. Plants are nature's green chemists: learning how plants make useful chemicals could help us to make our own plant-inspired chemicals in an environmentally friendly manner.

Using a synthesis of genomics, enzymology and phylogenetics, we will elucidate the steps taken in the evolution of a new specialised metabolic pathway in plants. In doing so we will provide insight into the interplay between enzyme and genome evolution. The model pathway we will investigate is Nepeta nepetalactone biosynthesis. The first step is to obtain high quality genome sequences of multiple Nepeta species and close relatives. These will be sequenced using Oxford Nanopore and Illumina technologies, and the metabolite profile of the plants will be assessed. In total we will obtain over eight genomes of closely related plants. Phylogenomic analyses will be conducted including species trees along with phylograms and chronograms of genes of interest. Comparative genomics will reveal syntenic relationships between genomes including conserved gene clusters.

The NEPS (nepetalactone related short chain dehydrogenases/reductases) family of enzymes have evolved from a single dehydrogenase ancestor into at least five subtypes with diverse dehydrogenase and [4+2]-cyclase activities. We will use ancestral sequence reconstruction to infer ancestral NEPS sequences, and then characterise the enzymes in order to discover how the NEPS gained their diverse activities. We will then use ancestral sequence reconstructions to resurrect ancestors of multiple genes involved in the nepetalactone biosynthesis pathway at specific points in evolutionary history. We will test multiple ancestral enzymes in multi-enzyme cascades, essentially resurrecting an ancestral metabolic pathway. This will provide an experimental exploration of how nepetalactone biosynthesis evolved. The biochemical data will be combined with inferred ancestral genomes to investigate how enzyme evolution and genome evolution interact in the emergence of new chemistry in plants.